Neutrophil Effector Responses Research Articles

MARCKS Inhibition Alters Bovine Neutrophil Responses to Salmonella Typhimurium.

Neutrophils are innate immune cells that respond quickly to sites of bacterial infection and play an essential role in host defense. Interestingly, some bacterial pathogens benefit from exuberant neutrophil inflammation. Salmonella is one such pathogen that can utilize the toxic mediators released by neutrophils to colonize the intestine and cause enterocolitis. Because neutrophils can aid gut colonization during Salmonella infection, neutrophils represent a potential host-directed therapeutic target. Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin-binding protein that plays an essential role in many neutrophil effector responses. We hypothesized that inhibition of MARCKS protein would alter bovine neutrophil responses to Salmonella Typhimurium (STm) ex vivo. We used a MARCKS inhibitor peptide to investigate the role of MARCKS in neutrophil responses to STm. This study demonstrates that MARCKS inhibition attenuated STm-induced neutrophil adhesion and chemotaxis. Interestingly, MARCKS inhibition also enhanced neutrophil phagocytosis and respiratory burst in response to STm. This is the first report describing the role of MARCKS protein in neutrophil antibacterial responses.

Human Neutrophil Response to Pseudomonas Bacteriophage PAK_P1, a Therapeutic Candidate.

The immune system offers several mechanisms of response to harmful microbes that invade the human body. As a first line of defense, neutrophils can remove pathogens by phagocytosis, inactivate them by the release of reactive oxygen species (ROS) or immobilize them by neutrophil extracellular traps (NETs). Although recent studies have shown that bacteriophages (phages) make up a large portion of human microbiomes and are currently being explored as antibacterial therapeutics, neutrophilic responses to phages are still elusive. Here, we show that exposure of isolated human resting neutrophils to a high concentration of the Pseudomonas phage PAK_P1 led to a 2-fold increase in interleukin-8 (IL-8) secretion. Importantly, phage exposure did not induce neutrophil apoptosis or necrosis and did not further affect activation marker expression, oxidative burst, and NETs formation. Similarly, inflammatory stimuli-activated neutrophil effector responses were unaffected by phage exposure. Our work suggests that phages are unlikely to inadvertently cause excessive neutrophil responses that could damage tissues and worsen disease. Because IL-8 functions as a chemoattractant, directing immune cells to sites of infection and inflammation, phage-stimulated IL-8 production may modulate some host immune responses.

Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis.

Rac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens. Here, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control. All the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis. Collectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils.

The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses.

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating β2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and β2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs.

Distinct transcription factor networks control neutrophil-driven inflammation.

SummaryNeutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We show active chromatin remodelling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses, and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Our study therefore represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.

β2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of β2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of β2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two β2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting β2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.

Variation in Neutrophil Function Among Healthy Subjects Influences Response to Bacteria

Introduction: We have recently shown that healthy subjects have variable neutrophil degranulation responses to antigen/antibody immune complexes (ICs; Duarte ME Blood 2019). With repeated testing over 2 years, we have shown that susceptibility to neutrophil activation represents a fixed phenotype: some healthy subjects have neutrophils which robustly degranulate in response to ICs, while others have neutrophils that are minimally reactive (>30-fold variation in release of MMP9, representative of tertiary granules). Whether this variation in degranulation responses is specific to ICs or is applicable to other stimulants is not known. Because neutrophils are involved in the first steps of host defense against bacterial pathogens, we aimed to determine if these phenotypes are associated with differences in neutrophil effector functions which may contribute to host defense. Methods/Results: To determine if observed neutrophil phenotypes are relevant to host defense, we compared neutrophil effector responses from "high" and "low" donors after being challenged with bacterial products including the bacterial tripeptide N-formyl-met-leu-phe (fMLP), LPS, and Staphylococcus aureus (USA300) supernatant. As a model IC, we chose to use KKO-platelet factor 4/heparin ICs, as neutrophil responses to this IC have been previously characterized by our lab. As a first step, we examined several healthy subjects and compared degranulation responses to ICs, fMLP, and LPS to determine if susceptibility to degranulation was preserved. As shown in Figure 1, the quantity of MMP9 released in response to ICs was highly correlated with MMP9 released in response to fMLP (Fig 1A) and to LPS (Fig 1B, r = 0.9 for both). We then determined if neutrophil phenotypes were preserved in response to bacteria. As shown in Figure 2, neutrophils from "high" and "low" donors differentially degranulated in response to Staphylococcus aureus supernatant, with continued preservation of the neutrophil phenotype. We next aimed to determine if the neutrophil phenotype was associated with differences in other neutrophil effector functions, besides degranulation, which are relevant to pathogen defense. First, we focused on neutrophil extracellular trap (NET) release, which has an important role in host defense. As shown in Figure 3, "high" and "low" donors differed in NET release in response to ICs, as quantified by visualization using immunofluorescent microscopy. This finding is supported by our previous report that neutrophil phenotypes are correlated with differences in MPO release, a NET component (Duarte ME, et al 2019). We also examined reactive oxygen species (ROS) generation, a primary mechanism by which neutrophils eliminate pathogens. As shown in Figure 4, using DCFH-DA, a fluorescent probe, we show that "high" and "low" donors differ in ROS generation in response to PMA and fMLP. Conclusions: Taken together, our studies demonstrate that neutrophil responses to a variety of stimuli (ICs, LPS, and fMLP) are highly concordant among healthy donors with "high" or "low" neutrophil phenotypes and that these differences in neutrophil effector functions are likely relevant to host defense. Further studies are needed to determine how differences in innate neutrophil function may affect clinical outcomes during bacterial infection. Disclosures Arepally: Apotex: Consultancy, Research Funding; Biokit: Consultancy, Patents & Royalties; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Annexon Biosciences: Consultancy, Other; Alexion: Other; Novartis: Consultancy.

Importance of Fc Receptor γ-Chain ITAM Tyrosines in Neutrophil Activation and in vivo Autoimmune Arthritis.

Activating Fcγ receptors associated with Fc receptor γ-chain (FcRγ) are critical for mediating neutrophil effector functions in immune complex-mediated autoimmune diseases. FcRγ contains ITAM tyrosines and the in vivo role of these tyrosines has not been defined in neutrophils and arthritis. In this study, the in vivo functions of FcRγ ITAM tyrosines were characterized using wild type and ITAM tyrosine mutant (Y65F/Y76F) transgenic mice crossed to an FcRγ-deficient genetic background. FcRγ-deficient neutrophils showed undetectable cell surface expression of the activating Fcγ receptor IV, defective immune complex-induced superoxide production, degranulation and spreading. Although the re-expression of both the wild type and the ITAM tyrosine mutant (Y65F/Y76F) FcRγ could restore activating Fcγ receptor expression of FcRγ-deficient neutrophils, only the wild type transgenic form could mediate Fcγ receptor-dependent effector functions. In contrast, neutrophils carrying ITAM tyrosine mutant FcRγ were unable to produce superoxide, mediate degranulation and perform active spreading. In addition, our results confirmed the protection of FcRγ-deficient mice from autoimmune arthritis. Importantly, the presence of the wild type FcRγ transgene, in contrast to the ITAM tyrosine mutant transgene, partially reversed autoimmune arthritis development. The reversing effect of the wild type transgene was even more robust when animals carried the wild type transgene in a homozygous form. Collectively, FcRγ ITAM tyrosines play a critical role in the induction of neutrophil effector responses, the initiation and progression of an autoantibody-induced experimental arthritis in vivo, indicating a signaling, rather than just a receptor stabilizing function of the molecule.

The roles of NADPH oxidase in modulating neutrophil effector responses.

Neutrophils are phagocytic innate immune cells essential for killing bacteria via activation of a wide variety of effector responses and generation of large amounts of reactive oxygen species (ROS). Majority of the ROS in neutrophils is generated by activation of the superoxide-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Independent of their anti-microbial function, NADPH oxidase-derived ROS have emerged as key regulators of host immune responses and neutrophilic inflammation. Data from patients with inherited defects in the NADPH oxidase subunit alleles that ablate its enzyme function as well as mouse models demonstrate profound dysregulation of host inflammatory responses, neutrophil hyper-activation and tissue damage in response to microbial ligands or tissue trauma. A large body of literature now demonstrates how oxidants function as essential signaling molecules that are essential for the regulation of neutrophil responses during priming, degranulation, neutrophil extracellular trap formation, and apoptosis, independent of their role in microbial killing. In this review we summarize how NADPH oxidase-derived oxidants modulate neutrophil function in a cell intrinsic manner and regulate host inflammatory responses. In addition, we summarize studies that have elucidated possible roles of oxidants in neutrophilic responses within the oral mucosa and periodontal disease.

ICAM-1–expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia

AbstractIntracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1–deficient mice were defective in these effector functions. Mechanistically, ICAM-1–mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense.

Neutrophil effector responses are fully suppressed by secretory phospholipase A2-modified HDL

Neutrophil effector responses are fully suppressed by secretory phospholipase A2-modified HDL

Neutrophil effector responses are suppressed by secretory phospholipase A2 modified HDL

Neutrophil effector responses are suppressed by secretory phospholipase A2 modified HDL

Neutrophil effector responses are suppressed by secretory phospholipase A2 modified HDL

Secretory phospholipase A2 (sPLA2) generates bioactive lysophospholipids implicated in acute and chronic inflammation, but the pathophysiologic role of sPLA2 is poorly understood. Given that high-density lipoprotein (HDL) is the major substrate for sPLA2 in plasma, we investigated the effects of sPLA2-mediated modification of HDL (sPLA2-HDL) on neutrophil function, an essential arm of the innate immune response and atherosclerosis.Treatment of neutrophils with sPLA2-HDL rapidly prevented agonist-induced neutrophil activation, including shape change, neutrophil extracellular trap formation, CD11b activation, adhesion under flow and migration of neutrophils. The cholesterol-mobilizing activity of sPLA2-HDL was markedly increased when compared to native HDL, promoting a significant reduction of cholesterol-rich signaling microdomains integral to cellular signaling pathways. Moreover, sPLA2-HDL effectively suppressed agonist-induced rise in intracellular Ca2+ levels. Native HDL showed no significant effects and removing lysophospholipids from sPLA2-HDL abolished all anti-inflammatory activities.Overall, our studies suggest that the increased cholesterol-mobilizing activity of sPLA2-HDL and suppression of rise in intracellular Ca2+ levels are likely mechanism that counteracts agonist-induced activation of neutrophils. These counterintuitive findings imply that neutrophil trafficking and effector responses are altered by sPLA2-HDL during inflammatory conditions.

The function of neutrophils in sepsis

Neutrophils are an essential arm of the innate immune response. In patients with sepsis, reprogramming of neutrophil occurs, manifest by impaired recruitment of neutrophils to sites of infection, abnormal accumulation of neutrophils to remote sites, and dysregulation of neutrophil effector responses. This review examines the mechanisms underlying dysregulated neutrophil trafficking and function during sepsis. Mechanisms governing neutrophil function in sepsis are complex. Bacterial products, cytokines/chemokines, leukotrienes, and immunomodulatory hormones can modulate neutrophil migratory responses during sepsis via induction of cytoskeletal changes, disruption of polymorphonuclear leukocyte (PMN)-endothelial cell interactions, and alterations in G-protein-coupled receptor expression or signaling. Impaired chemotactic responses and alterations in neutrophil function can occur as a result of dysregulated PMN G-protein-coupled receptor and Toll-like receptor expression and/or signaling. As sepsis evolves, neutrophil gene expression is altered, leading to suppression of proinflammatory and immunomodulatory genes, as well as decreased production of reactive oxygen species. Neutrophil extracellular traps are produced to contain and kill invading pathogens, but can paradoxically promote further tissue damage. Neutrophil migration is a coordinated process that is altered at multiple stages during sepsis. In combination with impaired neutrophil function, these alterations culminate in defective innate immunity in septic patients. Defining the mechanisms involved and strategies to interrupt these deleterious responses requires further investigation.

INFLAMMATORY/CARDIOVASCULAR-METABOLIC RESPONSES IN A RAT MODEL OF BURN INJURY WITH SUPERIMPOSED INFECTION

Infection remains the major cause of morbidity and mortality in burn patients. Furthermore, the use of antibiotics in such patients has led to the prevalence of antibiotic-resistant microbial infections; one such infection in intensive care unit turns out to be caused by the enterococcal organisms. Our laboratory studies have used a rat model of bum injury and Enterococcus faecalis infection. Sprague-Dawley male rats ( approximately 250 g) were initially given an intragastric gavage of the antibiotic ciprofloxacin for 3 days. This procedure allowed for decontamination of intestine of gram-negative and some gram-positive organisms. The remainders of the gram-positive organisms were, to a large extent, Enterococci. After the decontamination procedure, rats were intra-abdominally inoculated with E. faecalis; inoculation involved preparation of sterilized rat fecal pellets impregnated with E. faecalis (10 colony-forming units) and their implants through a midline abdominal wall incision. Some of the rats that were implanted with the fecal pellets were subject to full-thickness skin bums ( approximately 30% total body surface area; 95 degrees C water for 7 s). Sham abdominal infection rats received a sterile pellet only; sham bum procedure consisted of exposing the skin to room temperature water. All sham and bum and/or E. faecalis infection procedures were carried out on rats under pentobarbital anesthesia. Inflammation and innate host defense-related responses were assessed via measurements of neutrophil effector responses, i.e., oxygen anion free radical (O2)/eIastase production, CD11b/CD18 expression, apoptosis, and tissue infiltration. Determining epithelial lactulose permeability, microvascular albumin leakage, and epithelial tight junction integrity assessed the status of intestinal function/structural derangements. The animals' metabolic and cardiovascular integrity was evaluated determining blood pH, p02, pC02, heart rate, respiratory rate, blood pressure, and cardiac output. Whereas the aforementioned measurements were carried out at 24 to 48 h postbum injury with and without the Enterococcal infection, animal mortality was determined for up to 5 days after the experimental injuries. The results of the studies indicated that whereas bum or E. faecalis infection alone did not produce significant mortality, the dual insult with bum and E. faecalis infection resulted in significant animal death accompanied by relatively more profound metabolic and cardiovascular derangements. Inappropriately heightened neutrophil effector responses were present with bum alone as well as with the dual bum and infection complications. These studies suggest that animal models of bum injury with Enterococcal infection complications simulate the adverse outcomes bum patients infected with Enterococcal organisms.